Characterization of DNA synthesis at a restrictive temperature in the temperature-sensitive mutants, tsFT5 cells, that belong to the complementation group of ts85 cells containing a thermolabile ubiquitin-activating enzyme E1. Involvement of the ubiquitin-conjugating system in DNA replication

A temperature-sensitive mutant defective in DNA replication, tsFT5, has been isolated from the mouse mammary carcinoma cell line FM3A. DNA synthesis in tsFT5 cells at a restrictive temperature (39 degrees C) has been characterized in detail. Incorporation of [3H]thymidine decreased rapidly after an...

Full description

Saved in:
Bibliographic Details
Published in:The Journal of biological chemistry 1993-08, Vol.268 (22), p.16803-16809
Main Authors: MORI, M, EKI, T, TAKAHASHI-KUDO, M, HANAOKA, F, UI, M, ENOMOTO, T
Format: Article
Language:English
Subjects:
Animals
Autoradiography
Biological and medical sciences
Cell Cycle - genetics
Cell Cycle - physiology
Cell Division
DNA Replication
Fundamental and applied biological sciences. Psychology
Genetic Complementation Test
Histones - metabolism
Ligases - metabolism
Mice
Mice, Inbred C3H
Molecular and cellular biology
Molecular genetics
Mutation
Phosphorylation
Replication
Temperature
Tumor Cells, Cultured
Ubiquitin-Activating Enzymes
Ubiquitin-Protein Ligases
Citations: Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:A temperature-sensitive mutant defective in DNA replication, tsFT5, has been isolated from the mouse mammary carcinoma cell line FM3A. DNA synthesis in tsFT5 cells at a restrictive temperature (39 degrees C) has been characterized in detail. Incorporation of [3H]thymidine decreased rapidly after an increase in temperature to 39 degrees C and the incorporation was less than 20% and 10% of the initial level after 4 and 8 h, respectively. Analysis by DNA fiber autoradiography revealed that the initiation of DNA replication at the origin of the replicons was impaired in tsFT5 cells but that the DNA chain elongation rate of the mutant cells did not decrease at the nonpermissive temperature. tsFT5 cells were confirmed to belong to the complementation group which includes ts85 cells arrested mainly in the G2 phase at the nonpermissive temperature. It has been observed that the amount of ubiquintin-conjugated histone H2A (uH2A) in ts85 cells decreases at the nonpermissive temperature (Marunouchi, T., Yasuda, H., Matsumoto, Y., and Yamada, M. (1980) Biochem. Biophys. Res. Commun. 95, 126-131). The amount of uH2A in tsFT5 cells also decreased rapidly at 39 degrees C. This decrease occurred at the same time as or slightly preceding to reduction in DNA synthesis, and the reappearance of uH2A was followed by the restoration of DNA synthesis after the temperature was reduced. A similar temporal relationship between decrease in the amount of uH2A and reduction in DNA synthesis was observed in ts85 cells cultured at 39 degrees C. However, the rates of the decrease of uH2A and of the reduction in DNA synthesis in ts85 cells were slower than those observed in tsFT5 cells. A comparison of the thermolability of purified ubiquitin-activating enzyme E1s revealed that the E1 from ts85 cells had a thermolability intermediate between those of the E1 from tsFT5 cells and of the wild-type cells. A reduction in the phosphorylation of histone H1 was observed in tsFT5 cells cultured at 39 degrees C, but the reduction occurred several hours after the decrease in uH2A and the reduction in DNA synthesis.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(19)85487-X